An approach to delineate primers for a group of poorly conserved sequences incorporating the common motif region

Glutathione synthetase (gshB) has previously been reported to confer tolerance to acidic soil condition in Rhizobium species. Cloning the gene coding for this enzyme necessitates the designing of proper primer sets which in turn depends on the identification of high quality sequence similarity in multiple global alignments. In this experiment, a group of homologous gene sequences related to gshB gene (accession no: gi-86355669:327589-328536) of Rhizobium etli CFN 42, were extracted from NCBI nucleotide sequence databases using BLASTN and were analyzed for designing degenerate primers. However, the T-coffee multiple global alignment results did not show any block of conserved region for the above sequence set to design the primers. Therefore, we attempted to identify the location of common motif region based on multiple local alignments employing the MEME algorithm supported with MAST and Primer3. The results revealed some common motif regions that enabled us to design the primer sets for related gshB gene sequences. The result will be validated in wet lab.     

Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth

Infection of the genitourinary tract with Group B Streptococcus (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death.     

Ammonia flux,physiological parameters,and <Emphasis Type="Italic">Symbiodinium</Emphasis> diversity in the anemonefish symbiosis on Red Sea coral reefs

Despite the ecological importance of anemonefish symbioses, little is known about how nutritional contributions from anemonefish interact with sea anemone physiology and Symbiodinium (endosymbiotic dinoflagellate) genetic identity under field conditions. On Red Sea coral reefs, we measured variation in ammonia concentrations near anemones, excretion rates of anemonefish, physiological parameters of anemones and Symbiodinium, and genetic identity of Symbiodinium within anemones. Ammonia concentrations among anemone tentacles were up to 49% above background levels, and anemonefish excreted ammonia significantly more rapidly after diurnal feeding than they did after nocturnal rest, similar to their excretion patterns under laboratory conditions. Levels of 4 physiological parameters (anemone protein content, and Symbiodinium abundance, chlorophyll a concentration, and division rate) were similar to those known for laboratory-cultured anemones, and in the field did not depend on the number of anemonefish per anemone or depth below sea surface. Symbiodinium abundance varied significantly with irradiance in the shaded reef microhabitats occupied by anemones. Most anemones at all depths harbored a novel Symbiodinium 18S rDNA variant within internal transcribed spacer region 2 (ITS2) type C1, while the rest hosted known ITS2 type C1. Association with Symbiodinium Clade C is consistent with the symbiotic pattern of these anemones on other Indo-Pacific reefs, but the C1 variant of Symbiodinium identified here has not been described previously. We conclude that in the field, anemonefish excrete ammonia at rapid rates that correlate with elevated concentrations among host anemone tentacles. Limited natural variation in anemonefish abundance may contribute to consistently high levels of physiological parameters in both anemones and Symbiodinium, in contrast to laboratory manipulations where removal of fish causes anemones to shrink and Symbiodinium to become less abundant.     

Interaction of thiocyanate with horseradish peroxidase. 1H and 15N nuclear magnetic resonance studies

Interaction of thiocyanate with horseradish peroxidase (HRP) was investigated by relaxation rate measurements (at 50.68 MHz) of the 15N resonance of thiocyanate nitrogen and by following the hyperfine shifted ring methyl proton resonances (at 500 MHz) of the heme group of SCN-.HRP solutions. At pH 4.0, the apparent dissociation constant (KD) for thiocyanate binding to HRP was deduced to be 158 mM from the relaxation rate measurements. Chemical shift changes of 1- and 8-ring methyl proton resonances in the presence of various amounts of thiocyanate at pH 4.0 yielded KD values of 166 and 136 mM, respectively. From the pH dependence of KD and the 15N resonance line width, it was observed that thiocyanate binds to HRP only under acidic conditions (pH less than 6). The binding was found to be facilitated by protonation of an acid group on the enzyme with pKa 4.0. The pH dependence of the 15N line width as well as the apparent dissociation constant were quantitatively analyzed on the basis of a reaction scheme in which thiocyanate in deprotonated ionic form binds to the enzyme in protonated acidic form. The KD for thiocyanate binding to HRP was also evaluated in the presence of an excess of exogenous substrates such as resorcinol, cyanide, and iodide ions. It was found that the presence of cyanide (which binds to heme iron at the sixth coordination position) and resorcinol did not have any effect on the binding of thiocyanate, indicating that the binding site of the thiocyanate ion is located away from the ferric center as well as from the aromatic donor binding site. The KD in the presence of iodide, however, showed that iodide competes with thiocyanate for binding at the same site. The distance of the bound thiocyanate ion from the ferric center was deduced from the 15N relaxation time measurements and was found to be a 6.8 A. From the distance as well as the change in the chemical shifts and line width of 1- and 8-methyl proton resonances, it is suggested that the binding site of thiocyanate may be located near heme, placed symmetrically with respect to 1- and 8-methyl groups of the heme of HRP. Similarity in the modes of binding of iodide and thiocyanate suggests that the oxidation of thiocyanate ion by H2O2 may also proceed via the two-electron transfer pathway under acidic conditions, as is the case for iodide.     

Disturbed brain phospholipid and docosahexaenoic acid metabolism in calcium-independent phospholipase A2-VIA (iPLA2β)-knockout mice

Calcium-independent phospholipase A₂ group VIA (iPLA₂β) releases docosahexaenoic acid (DHA) from phospholipids in vitro. Mutations in the iPLA₂β gene, PLA2G6, are associated with dystonia-parkinsonism and infantile neuroaxonal dystrophy. To understand the role of iPLA₂β in brain, we applied our in vivo kinetic method using radiolabeled DHA in 4 to 5-month-old wild type (iPLA₂β^+/+) and knockout (iPLA₂β^−/−) mice, and measured brain DHA kinetics, lipid concentrations, and expression of PLA₂, cyclooxygenase (COX), and lipoxygenase (LOX) enzymes. Compared to iPLA₂β^+/+ mice, iPLA₂β^−/− mice showed decreased rates of incorporation of unesterified DHA from plasma into brain phospholipids, reduced concentrations of several fatty acids (including DHA) esterified in ethanolamine- and serine-glycerophospholipids, and increased lysophospholipid fatty acid concentrations. DHA turnover in brain phospholipids did not differ between genotypes. In iPLA₂β^−/− mice, brain levels of iPLA₂β mRNA, protein, and activity were decreased, as was the iPLA₂γ (Group VIB PLA₂) mRNA level, while levels of secretory sPLA₂-V mRNA, protein, and activity and cytosolic cPLA₂-IVA mRNA were increased. Levels of COX-1 protein were decreased in brain, while COX-2 protein and mRNA were increased. Levels of 5-, 12-, and 15-LOX proteins did not differ significantly between genotypes. Thus, a genetic iPLA₂β deficiency in mice is associated with reduced DHA metabolism, profound changes in lipid-metabolizing enzyme expression (demonstrating lack of redundancy) and of phospholipid fatty acid content of brain (particularly of DHA), which may be relevant to neurologic abnormalities in humans with PLA2G6 mutations.     

Photosynthetic circadian rhythmicity patterns of Symbiodium,the coral endosymbiotic algae

Biological clocks are self-sustained endogenous timers that enable organisms (from cyanobacteria to humans) to anticipate daily environmental rhythms, and adjust their physiology and behaviour accordingly. Symbiotic corals play a central role in the creation of biologically rich ecosystems based on mutualistic symbioses between the invertebrate coral and dinoflagellate protists from the genus Symbiodinium. In this study, we experimentally establish that Symbiodinium photosynthesis, both as a free-living unicellular algae and as part of the symbiotic association with the coral Stylophora pistillata, is ‘wired’ to the circadian clock mechanism with a ‘free-run’ cycle close to 24 h. Associated photosynthetic pigments also showed rhythmicity under light/dark conditions and under constant light conditions, while the expression of the oxygen-evolving enhancer 1 gene (within photosystem II) coincided with photosynthetically evolved oxygen in Symbiodinium cultures. Thus, circadian regulation of the Symbiodinium photosynthesis is, however, complicated as being linked to the coral/host that have probably profound physiochemical influence on the intracellular environment. The temporal patterns of photosynthesis demonstrated here highlight the physiological complexity and interdependence of the algae circadian clock associated in this symbiosis and the plasticity of algae regulatory mechanisms downstream of the circadian clock.     

Ontogenetic variations in the venom proteome of the Amazonian snake Bothrops atrox

Background  

Bothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Previous studies have demonstrated that the biological and pharmacological activities of B. atrox venom alter with the age of the animal. Here, we present a comparative proteome analysis of B. atrox venom collected from specimens of three different stages of maturation: juveniles, sub-adults and adults.     

Analysis of intrasugar interproton NOESY cross-peaks as an aid to determine sugar geometries in DNA fragments

A systematic analysis of the conformation of deoxyribofuranose rings in DNA fragments has been described using two-dimensional nuclear Overhauser effect spectroscopy (2D NOESY). The approach is based on the interpretation of the intrasugar proton-proton distances which can be estimated using a low-mixing-time pure-absorption mode w1-scaled NOESY spectrum. The experimental distances are compared with the theoretical values calculated as a function of pseudorotation phase angle (P) describing the sugar geometries. The approach can be used as a complementary aid to J couplings for establishing sugar conformations in individual nucleotide units of DNA fragments. Using this strategy on d-ACATCGATGT, we observed that individual nucleotides exhibit O4'-endo sugar pucker. The results rule out possibilities of the existence of a fast equilibrium (on the NMR time scale) between C2'-endo (or S-domain) and C3'-endo (or N-domain) sugar puckers.